Treatment of obesity, the metabolic syndrome, type 2 diabetes, cardiovascular diseases, dementia, alzheimer&#39;s disease and inflammatory

ABSTRACT

A product for use in the treatment of obesity, the metabolic syndrome, type 2 diabetes, cardiovascular diseases, dementia, alzheimers disease and inflammatory bowel disease comprising at least one isolated bacterial strain from the species  Prevotellaceae , wherein the strain is selected from the group consisting of  Prevotella copri, Prevotella stercorea, Prevotella histicola, Prevotella ruminicola, Prevotella Bryantii  25A and  Prevotella distasonis . The product may be a food product.

FIELD OF INVENTION

A product for use in the treatment of obesity, the metabolic syndrome,type 2 diabetes, cardiovascular diseases, dementia, alzheimers diseaseand inflammatory bowel disease comprising at least one isolatedbacterial strain from the species Prevotellaceae, herein the strain isselected from the group consisting of Prevotella copri, Prevotellastercorea, Prevotella histicola, Prevotella ruminicola, PrevotellaBryantii 25A and Prevotella distasonis. The product may be a foodproduct.

BACKGROUND OF INVENTION

Many food—and ingredient companies within the field have increased theiractivities to identify ingredients as well as food products which uponuse will provide beneficial health effects on the consumer in additionto providing essential nutrients, so called functional foods. Examplesare viable microorganisms, so called probiotics which have shownspecific health benefits on gut health and which are included in foodproduct e.g. different dairy products such fermented milk, yoghurt, aswell as different beverages. Other examples of food products with healthbenefits are foods containing dietary fibre (DF), prebioticcarbohydrates, stannol, omega-3 fatty acids, vitamins, polyphenols, lowglycaemic impact foods, etc.

From the public health perspective there is currently a particular needto develop new food products that could act to improve glucosemetabolism and reduce obesity and related disorders. Impairment ofglucose metabolism is also associated with impaired cognitivefunctioning. The prevalence of life style related disorders such asobesity and type 2 diabetes (T2D) is increasing globally, and it hasbeen proposed that the number of people suffering from T2D worldwidewill increase from presently 366 million to reach 552 million by year2030. The need for preventive strategies is thus urgent. Diet basedprevention is recognized as the most efficient strategy in the combat oflife style related disease, and epidemiological studies support thate.g. high intake of whole grain foods and legumes is beneficial in theprevention and management of diabetes, and for weight control.

Obesity is a major factor contributing to cardio-metabolic disorders,but the underlying mechanisms are not fully known. However, a keyfeature appears to be “metabolic inflammation” and activation of theinnate system. Dietary patterns for example high-GI food and energydense food, are increasingly being considered predictive of future riskof cardiovascular diseases. Today, there is a growing body of knowledgein support of the importance of a healthy gut microbiota in the combatof cardiometabolic disorders and the gut eco-system is beingacknowledged as modulator of host metabolism, appetite—and weightregulation. The metabolic “crosstalk” between the gut microbiota andperipheral tissues has been suggested to be regulated through gutfermentation of indigestible dietary components, such as indigestiblecarbohydrates.

Much of the evidence regarding the role of fermentable gut substrates onhost metabolism stem from studies with inulin showing benefits onglucose metabolism and weight regulation in animal experimental models.

Recently the importance of the gut microbiota was documented in studiesbased on faecal transplantation from lean human donors to subjectssuffering from the metabolic syndrome (MetS); the MetS representing acluster of risk factors identifying subjects at high risk of developingT2D and cardiovascular disease. The faecal transplantation resulted inincreased insulin sensitivity in the T2D subjects 6 weeks after faecalinfusion. These new findings are exciting and add to the knowledgeregarding the importance of a healthy microbiota. However, faecaltransplantations may be both complicated and risky.

Thus there is a need for strategies to increase the number of beneficialbacteria, and promote a healthy balance of the gut microbial compositionby other means. Up to date most studies demonstrating increases ofbeneficial bacteria following diet manipulation are cross sectional andthus do not demonstrate causality. This can be achieved by supplyingviable bacteria through enemas, or through food products, including socalled probiotics, or by dietary supply of sufficient amounts of colonicsubstrates e.g. specific prebiotic substrates in the habitual diet, orby combining pro- and prebiotics.

Despite the efforts in the food industry to develop new food productswith improved health properties or functional food type products thereis still huge problem with obesity as well as an increasing populationof subjects with MetS or T2D. These conditions are typically treatedwith drugs. However, if healthy subjects or people at risk of diseasecould be offered food products specifically designed to counter-actearly disease processes, this would prevent development of obesity andMetS, including impairment of cognitive functioning. Moreover, such foodproducts could also facilitate disease management in patients withmanifest disease related to MetS.

SUMMARY OF THE INVENTION

The invention relates to the unique finding that species belonging toPrevotellaceae have beneficial effects on host health. The speciesbelonging to Prevotellaceae can be used in any kind of food products, oras an ingredient in a food product, as a probiotic with or without addedindigestible carbohydrates, administrated enclosed in a capsule, as anenema or a suppository to improve the health of the consumer.

In a first aspect the invention relates to a product for use in thetreatment of obesity, the metabolic syndrome, type 2 diabetes,cardiovascular diseases, dementia, alzheimers disease and inflammatorybowel disease comprising at least one isolated bacterial strain from thespecies Prevotellaceae, wherein the strain is selected from the groupconsisting of Prevotella copri, Prevotella stercorea, Prevotellahisticola, Prevotella ruminicola, Prevotella Bryantii 25A and Prevotelladistasonis. By such a product it is for the first time possible toprovide new healthy food ingredients as well as food products, capsules,enemas or suppositories which could be useful in the treatment of anumber of diseases mentioned above to improve glucose metabolism andreduce risk factors in the metabolic syndrome. The product may beprovided together with one or more dietary fibres and/or resistantstarch.

Further advantages and objects with the present invention will bedescribed in more detail, inter alia with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Blood glucose—and serum insulin responses (incremental changes(Δ)) to the standardized breakfast after 3 days consumption of barleykernel based bread (BB) and white wheat bread (WB), respectively.Consumption of BB resulted in lower postprandial glucose—and insulinpeak concentrations (P<0.01, and P<0.001, respectively), and lowerglucose—and insulin areas under the curves (0-150 min: P<0.01 andP<0.001, respectively) as compared to the reference WB (n=39).

FIG. 2. Blood glucose—and serum insulin responses (incremental changes(Δ)) to the standardized breakfast after 3 days consumption of barleykernel based bread (BB) and white wheat bread (WB), respectively.Consumption of BB resulted in lower postprandial glucose—and insulinpeak concentrations (P<0.05 for both), and lower glucose—and insulinareas under the curves (0-150 min: P<0.05 and P<0.01, respectively) ascompared to the reference WB (n=20).

FIG. 3. Breath H2 excretion at the standardized breakfast, following 3days consumption of barley kernel based bread (BB) and white wheat bread(WB), respectively. Consumption of BB resulted in significantlyincreased breath H2 compared to the reference meal WB (meanconcentration during the experimental day, P<0.01 (n=20).

FIG. 4. Plasma PYY concentrations at the standardized breakfast,following 3 days consumption of barley kernel based bread (BB) and whitewheat bread (WB), respectively. Consumption of BB resulted insignificantly increased plasma concentrations of PYY (0-120 min),compared to three days intake of WB (main effect during the experimentalday, P<0.05 (n=20).

FIG. 5. Blood glucose responses in responders (n=10) and non-responders(n=10) after the standardized breakfast, following 3 days consumption ofbarley kernel based bread (BB) or white wheat bread (WB), respectively.In the responders group, three days consumption of BB resulted insignificantly lower glucose responses, compared to three days intake ofWB (area under the curve 0-150 min, P<0.0001). No improvements inglucose tolerance (area under the curve) of the BB were seen in thenon-responders group.

FIG. 6. Serum insulin responses in responders (n=10) and non-responders(n=10) after the standardized breakfast, following 3 days consumption ofbarley kernel based bread (BB) and white wheat bread (WB), respectively.In the responders group, three days consumption of BB resulted insignificantly lower insulin responses, compared to three days intake ofWB (area under the curve 0-120 min, P<0.01). No improvements of the BBwere seen in the non-responders group.

FIG. 7. Blood glucose responses in responders (n=7) and non-responders(n=7) after the standardized breakfast, following 3 days consumption ofbarley kernel based bread (BB) and white wheat bread (WB), respectively.In the responders group, three days consumption of BB resulted insignificantly lower glucose responses, compared to three days intake ofWB (area under the curve 0-150 min, P<0.0001). No improvements inglucose tolerance (area under the curve) of the BB were seen in thenon-responders group.

FIG. 8 shows the taxonomic variation of the major gut microbial speciesacross the different groups involved in the study, data has beengenerated after 454 pyrosequencing analysis. The six bars represent thegroups involved in the study: R-control (responder control), R-WB(responders white wheat bread), R-BB (responders barley kernel basedbread). NR=non responders. Control means that the samples have beencollected without consumption of test breads (BB or WB).

FIG. 9 shows that mice monocolonized with Prevotella copri (n=6) for twoweeks exhibit improved oral glucose tolerance and lower serum insulinlevels compared with mice mono-colonized with Bacteroidesthetaiotaomicron (n=6) (P=0.0076). Mice bi-colonized with P. copri andB. thetaiotaomicron (n=7) also show better oral glucose tolerance incomparison with the mono B. thetaiotaomicron colonized mice (P=0.0426).

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the context of the present application and invention, the followingdefinitions apply: The term “metabolic syndrome” or MetS is intended tomean the cluster of risk factors such as obesity, hyperlipidemia,hypertension and glucose intolerance, identifying subjects at high riskof developing type2 diabetes (T2D) and cardio-vascular disease. The term“probiotic” is intended to mean a viable microorganism, such as bacteriathat upon colonisation in the gut confer a health benefit on the host.

The term “dietary fibre” or “DF” is intended to mean carbohydratesincluding three or more monomers that resist digestion and absorption inthe small intestine, and are completely or partially fermented in thecolon by gut bacteria. In this application the term DF relates tonon-starch derived indigestible polysaccharides such as beta-glucans,arabinoxylans, cellulose, oligosaccharides, fructans, pectin, guar gum.Indigestible substrates that are closely associated with indigestiblepolysaccharides in the plant are also included in the definition of DF(e.g. indigestible protein fractions, phenolic compounds, lignin, waxes,phytates, phytosterols).

The term “resistant starch” (RS) is intended to mean starch—and starchdegradation products that escape digestion in the small intestine ofhealthy individuals, i.e. starch derived DF. RS can deliver some of thebenefits of insoluble DF and some of the benefits of soluble DF. RS mayderive from botanically encapsulated starch, ungelatinised—orretrograded starch.

The term “retrograded starch” is intended to mean re-crystallisation ofstarch molecules after cooking and cooling, resulting in structuresresistant to digestion and absorption in the small intestine.

The term “botanically encapsulated starch” is intended to mean starchthat is physically entrapped within the food matrix, or botanical cellsmaking it inaccessible to digestive enzymes in the small intestine.

The term “ungelatinised starch” is intended to mean unprocessed starchgranules that occurs in its natural form, such as starch in uncookedpotatoes or uncooked cereals, or starch granules that have resistedgelatinisation upon processing, resulting in a retained indigestibleintact crystalline structure.

The term “prebiotic” is intended to mean indigestible food components,preferentially indigestible carbohydrates such as DF and RS, thatstimulate the growth and/or activity of bacteria in the digestive systemin ways associated with health benefits.

The term “synbiotics” is intended to mean a combination of probiotic andprebiotic.

The term SCFA is intended to mean short chain fatty acids produced frommicrobial gut fermentation of indigestible carbohydrates.

The term “encapsulated” is intended to mean that the Prevotella strainmay be encapsulated to be protected from the environment (e.g. oxygenand acidity) and thus will be intact and retain the qualities andactivities in the food product and during passage through the gut. Thetechniques used for encapsulation may be based on e.g. alginate-, guargum-, xanthan gum-, locust bean gum-, carrageenan gum-, or other dietaryfibre,—casein-, prebiotics—or starch based edible films, pickeringemulsions etc. Different encapsulation technologies may be applied,which some are described by R Vidhyalakshmi et.al., 2009 [1].

The term “responders” is intended to mean human subjects that afterconsumption of the prebiotic barley kernel product received improvedglucose regulation.

The term “non-responders” refers to human subjects that afterconsumption of the prebiotic barley kernel product do not displayevidence of improved glucose regulation.

The term “enema” is used to depict a procedure by which a solutioncontaining the Prevotella spp is introduced with or without prebioticsubstrate to the gut through the rectum in order to beneficiallymodulate the gut microbial composition.

The term metabolic inflammation is used to depict: low-grade, chronicinflammation orchestrated by metabolic cells in response to excessnutrients and energy associated with insulin resistance and metabolicdysfunction.

The Invention

It has been found that intrinsic DF and RS in barley kernel productspossess beneficial effects with respect to cardio-metabolic risk markersand appetite regulation, which is probably the same for other fibregroups as well. Hence, glucose tolerance and insulin economy wasimproved in a time perspective of 11-14 h after intake of barley kernelproducts, which is in an over-night perspective from a barley kernelevening meal to a subsequent standardised breakfast in healthy subjects.

Another important finding was an increase in plasma concentrations ofGLP-1 and other appetite regulatory hormones, and a decrease involuntary energy intake, while simultaneously reducing perceived hunger.In addition, barley indigestible carbohydrates reduced markers ofmetabolic inflammation. A state of increased metabolic inflammation is arecognized cardiometabolic risk factor, making food concepts that reducemetabolic inflammation promising in prevention of the MetS and T2D.Increased production of breath hydrogen (marker of gut fermentation) andSCFA were seen, indicative of increased gut fermentation activity afterintake of barley indigestible carbohydrates. Significant associationswere found between markers of gut fermentation and improved glucoseregulation, demonstrating prebiotic mediated effects of barley kernelbased products on metabolic risk markers, i.e. the beneficial effectsseen are related to mechanisms derived from gut bacterial fermentationof indigestible carbohydrates. SCFA produced during gut microbiotafermentation provide energy to colonic enterocytes, and in additionSCFAs also function as signaling molecules.

Consequently, it has been demonstrated that SCFA produced by bacterialfermentation may trigger signaling cascades through acting on SCFAreceptors on L-cells, resulting in increased release of gut peptidessuch as GLP-1, and PYY (in vitro model). The results obtained clearlydemonstrate a prebiotic potential of DF and RS in cereal products suchas barley kernel products

Surprisingly, it has been found in healthy subjects that the benefitsobserved after barley kernel products (bread) regarding cardio-metabolicrisk markers and appetite regulation could be associated with a specificalteration of the gut microbiota, and we identified an increase inPrevotella species. Furthermore, the transfer of a specific Prevotellastrain (Prevotella copri) to germ free mice improved glucose tolerancein the animals. The results put forward a probiotic effect of theidentified bacterial strain with benefits on glucose metabolism. On eofthe criterias for selection of Prevotella strains was occurrence ofglycoside hydrolases, needed for the degradation of complex sugars. Allthe selected strains have glycoside hydrolases, and are able to degradea large range of complex sugars. An additional criterion was theselected strains to produce succinate as main products from thedegradation of the complex sugars. Examples of the strains werePrevotella copri, Prevotella stercorea, Prevotella histicola, Prevotellaruminicola, Prevotella Bryantii 25A and Prevotella distasonis. Mixturesof one or more of the different strains may also be possible, such as 2,3, 4, 5 or 6 different strains. The strain may be present in an amountof from 10⁷ or more such as 10⁷, 10⁸, 10⁹ or even to higher amounts.

In addition, the results for the first time reveal that the investigatedprebiotic substrates, i.e. DF and RS present in barley kernel products,in combination with the Prevotella spp. strain could be particularlyvaluable in a synbiotic approach since these gut substrates favoured anincrease in Prevotella. The results were obtained in middle aged/elderlyhealthy subjects at fasting and after a standardised breakfast after 3days ingestion of a barley kernel based product (based on 100 gavailable carbohydrates/day). The barley kernel product was comparedwith a white wheat bread reference product using a cross-over design. Byaffecting the gut micro-biota composition and increasing the proportionof Prevotella spp, the barley kernel based product resulted in increasedgut fermentation activity (breath hydrogen (H₂) as a test marker)(P<0.001), increased concentrations of SCFA (P<0.05), improved glucoseregulation (reduced glucose—and insulin concentrations (P<0.05),increased concentrations of PYY (a satiety hormone released in the gut)(P<0.05), increased fasting concentrations of GLP-1 (an anti-obesity andanti-diabetic gut hormone), increased perceived fasting satiety, andreduced fasting hunger sensations. By altering the gut-microbiotacomposition and increasing the Prevotella spp, intake of barley kernelbased food rich in barley DF and RS increased the gut fermentationmetabolite succinate in both humans and mice.

The results further revealed that some individuals (approximately 15%)among the test subjects did not achieve an improved glucose regulationfollowing intake of the barley kernel based test product (determined byglucose—and/or insulin response to a standardised meal after 3 daysintervention with barley). Interestingly, neither did these“non-responders” acquire increased gut Prevotella concentrations afteringestion of the barley kernel product. This emphasise the causalrelationship between the improved glycaemic regulation and increasedabundance of Prevotella spp which is entirely new knowledge in thepublic domain. Also, these results indicate for the first time thatcertain individuals do not alter their gut microbiota composition asreadily, and may particularly benefit from oral ingestion of theproposed combination of barley prebiotics (DF+RS) and Prevotella spp; orby Prevotella spp ingestion only.

By use of for example a barley kernel food product rich in DF and RS orDF or RS, benefits on blood glucose control, markers of metabolicinflammation, and appetite regulation have been established. Themetabolic benefits were associated with markers of gut fermentation,i.e. breath hydrogen, plasma SCFA which indicates a prebioticsmechanism.

Gut microbial mapping of faecal samples from healthy humans ingestingthe above mentioned barley kernel product revealed that the benefits onmetabolic risk markers and appetite regulation were linked to a specificalteration of the composition of microorganisms present in the gut.Subjects that ingested the above mentioned barley kernel food productshowed an increase in Prevotella species in the gut microbiota andimproved cardiometabolic risk markers as compared to subjects that didnot ingest the barley kernel food product (see the Examples below).

Further, it was shown that mono-colonisation of germ free mice with aPrevotella spp. strain improved glucose metabolism in the animals. Thus,the conclusion from the above results being that species related toPrevotella is suitable to be used as a probiotic with for exampleantidiabetic properties and weight regulating potential. The tightrelation between impairment of glucose metabolism and impaired cognitivefunction also make relevant use of Prevotella species for prevention ofcognitive decline associated with MetS. Other diseases or disorder arementioned above.

From the above experiments and conclusion the invention was identifiedto be related to a product, such as a food product or food ingredient orformulation to be used as an enemia, comprising at least one humanisolated bacterial strain from the Prevotellaceae. The bacterial may begenetically modified, such as modified to produce succinate. Examplesare Prevotella copri, Prevotella stercorea, Prevotella histicola,Prevotella ruminicola, Prevotella Bryantii 25A and Prevotelladistasonis.

The probiotic food product can be used alone, or in combination withother components, such as at least one type of dietary fibres, such asDF and RS or DF or RS and in some examples also succinic acid toregulate the glucose metabolism and reduce risk factors in the metabolicsyndrome in a mammal such as in humans. Examples of diseases ordisorders that would be treated includes treatment of obesity, themetabolic syndrome, type 2 diabetes, cardiovascular diseases, dementia,alzheimers disease and inflammatory bowel disease, such as obesity,metabolic syndrome and type 2 diabetes.

The probiotic food product may also comprise at least one DF and/or oneRS, natural or synthetic, purified, mixtures or variants thereof.Examples of DF and RS are DF and RS from cereals or legumes such asviscous and non-viscous DF from barley, rye, wheat, oats, legume seeds,beta-glucans, guar gum, lignin, lignans and oligosaccharides such asgalacto-oligosaccharides and fructo-oligosaccharides, botanicallyencapsulated RS, retrograded RS, chemically modified starch orungelatinised RS. The product may comprise at least one type ofresistant starch (RS), a natural one, synthetic, purified, mixtures orvariants thereof. Examples of RS are vegetable starch such asretrograded starch, botanically encapsulated starch, ungelatinizedstarch and cyklodextrins, or chemically modified starch. The fibre mayfor example be from barley kernels. The ratio of RS/DF could for examplebe 10.8 (RS)/13.04 (DF). The DF could be dietary fibre from barley,wheat, rye, oats or extracted beta-glucans, guar gum, lignin, lignansand oligosaccharides such as galacto-oligosaccharides andfructo-oligosaccharides, botanically encapsulated RS, retrograded RS,chemically modified starch or ungelatinised RS. The product may compriseat least one type of resistant starch (RS), a natural one, synthetic,purified, mixtures or variants thereof. Examples of RS are vegetablestarch such as retrograded starch, botanically encapsulated starch,ungelatinized starch and cyklodextrins, or chemically modified starch.The fibre may for example be from barley kernels.

The product may for example contain one or more bacterial straintogether with RS and/or DS to achieve the expected results.

In another embodiment the invented product comprises an additionalbacterial strain, wherein the said strain may produce succinate.Examples of such strains include any bacterial strain that is beneficialand includes e.g. Lactobacillus, such as Lactobacillus reuteri,Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillusplantarum, Roseburia and Bifidobacteria, such as Bifidobacterium longum,Bifidobacterium lactis, Bifidobacterium animalis and Bifidobacteriumbifidum. The additional bacterial strain may as well be geneticallymodified, such as to produce succinate.

The product may also contain succinate in the free form or added with asuccinate producing bacterial strain. A strain that produces succinatenaturally or a strain that has been genetically modified to producesuccinate.

Accordingly, the product may be capsulated or lyophilised which is wellknown for a person skilled in the art.

In another embodiment the bacterial strain from the Prevotellaceae isencapsulated in any form to protect the strain from oxygen. Examples ofencapsulation techniques includes edible films, stable Pickeringemulsions employing starch granules as barrier material, encapsulationbased on polysaccharides e.g. beta-glucans, guar gum, xanthan gum,locust bean gum, carrageenan gum-, prebiotics, alginate, milk proteinetc. Other techniques include inter-polymer complex formation insuper-critical carbon dioxide. Different encapsulation technologies maybe applied, which some are described by R Vidhyalakshmi et.al., 2009.

In another embodiment the bacterial strain/strains from Prevotellaceaecan be provided through enema alone or in a composition with prebioticsubstrates such as DF and/or RS.

The product may for example be any suitable food product and includesany food product that is not or mildly heat treated, or where thePrevotella can be introduced after heat-treatment, such as beverages,shots (e.g. fruit- or dairy based), smoothies, drinks, juices, tablewater, cold soups/soup mixes, oils products, spread, dressings, coldsauce/sauce mix, salsa, dairy products, ice-cream, and cereal basedbeverages, containing or supplemented with the Prevotella or thePrevotella and the prebiotic combined. The Prevotella may also beencapsulated and included in the foods described above, hence increasingresistance to environment and processing conditions.

The food product may also be heat-treated foods such as soft bread,crisp bread, flat-bread, tortillas, porridges, breakfast cereals, cerealbars or other snacks, potato powder or other instant food product, readyto eat meals, containing the prebiotic e.g. from barley, to be consumedwith or without supplementation with the Prevotella. Capsules containingthe Prevotella spp. to be ingested with the products listed above, orconsumed for provision of the Prevotella. The capsules may additionallybe made from prebiotic carbohydrates, e.g. from barley. The Prevotellamay also be encapsulated e.g. by stable emulsion techniques and includedin the foods.

The Prevotella spp. can also be provided as dry powder and packed indisposable containers to be added to meals, e.g. breakfast cereals. Thefood product can also be e.g. a drink, soup, yoghurt, cold cerealpudding or shot provided in a two chamber package, with Prevotella withor without prebiotic in one of the chambers and the food product asabove in the other, to be mixed upon consumption. The chamber withPrevotella may also consist of a straw, which is included with thepackage, or distributed separately. The Prevotella will then be consumedby drinking through the straws.

The product can be a stable emulsion, where the Prevotella with orwithout prebiotics are encapsulated in emulsion droplets. These productscan be administered as e.g. a shot.

In the case that the Prevotella are included in capsules, the capsulesshould preferably be in a form of entero capsules. Prevotella can alsobe included in entero tablets, depot tablets, depot (prolonged-release)capsules, or prolonged-release granules.

The product may also be a food ingredient that is to be added to a foodproduct.

The invention also defines a product that could be used in the treatmentof a number of diseases or disorders including those mentioned below. Itcould be used for the treatment of obesity, and associated disordersrelated to impaired blood glucose regulation such as type 2 diabetes aswell as protection against subclinical inflammation and associateddisease such as cardio-vascular disease. Additionally the protectedconcepts counteract features of the pathogenesis of dementia. Also,dysregulation of blood glucose is associated with inflammation andendothelial damage hence providing yet a link between the presentlyprotected concept and prevention of cardiovascular disease.

Consequently, even mild impairment of glucose regulation in the normalrange is related to a significantly lower performance in cognitive testsand the presently described concept improves gluco-regulation suggestingbenefits adjunct to dementia. Also the presently described protectedconcept stimulate incretin hormones such as GLP-1. GLP-1 exertneuroprotective and anti-apoptotic effects, reduce beta-amyloid (Aβ)plaque accumulation, modulate long-term potentiation and synapticplasticity, and promote differentiation of neuronal progenitor cells,hence preventing against dementia and Alzheimer's disease. Theneuroprotective effects of GLP-1: possible treatments for cognitivedeficits in individuals with mood disorders. Additionally thestimulation of GLP-2 observed with the protected concept protectsagainst inflammatory bowel disease, and it is known that exogenous GLP-2can protect the mucosa from chemotherapy-induced mucositis in rats.

The invention relates also to the identified product, such as a foodproduct or food ingredient and the use of that product to improveglucose metabolism, lower metabolic inflammation, and facilitateappetite regulation as well as reduce risk factors in the metabolicsyndrome. This could be useful in the prevention and regulation ofdisorder related to the MetS, such as obesity, glucose intolerance,diabetes or cardiovascular disease as well as in subject suffering fromimpairment of cognitive functioning related to the MetS. The inventedprobiotic may also be used in enema formulations or suppositories withor without prebiotic substrates.

The invention also relates to the use of the product as defined above toreduce risk factors in the metabolic syndrome, improve glucosemetabolism, facilitate weight regulation and reduce risk of cognitivedecline related to the MetS.

Finally, the invention relates to the use of succinate or a succinateproducing bacterial strain to improve glucose metabolism, facilitateweight regulation and reduce risk factors in the metabolic syndrome.

The product may be used for humans, horses as well as dogs and cats.

Following examples are intended to illustrate, but not to limit theinvention in any manner, shape, or form, either explicitly orimplicitly.

EXAMPLES Example 1 Prebiotic Carbohydrates in Barley Kernel BasedProducts Beneficially Affect Metabolic Risk Markers, Appetite RegulatoryHormones, and Perceived Satiety in Healthy Subjects

Study Intension and Summary of the Study Design

The aim of the study was to evaluate effects of intrinsic indigestiblecarbohydrates, DF and RS present in barley kernel based products on riskmarkers related to the MetS. Thirty-nine healthy middle-aged subject,aged 64.5±5.6 years, with normal body mass indices (mean±SD=23.6±2.3kg/m²) were provided a barley kernel based bread- or a white wheat bread(WB, reference product) during three consecutive days in a cross-overdesign. The following day (day four) a standardised breakfast was servedand physiological test markers determined at fasting and repeatedly inthe postprandial period (0-180 min). The study was divided in threesub-studies (STUDIES A-C). The physiological test markers in STUDY Aincluded determinations of blood glucose, serum insulin, breath hydrogen(H₂) excretion (marker of colonic fermentation), and registration ofsubjective appetite sensations. Venous blood was collected repeatedlyfor further evaluations in a sub-group of the cohort (see below). Inaddition, faecal samples were collected prior to start of the study andafter each intervention period for characterisation of the gutmicrobiota (STUDY A).

From the cohort described above, additional physiological test markerswere determined from 20 subjects, randomly chosen (64.1±5.9 years,23.5±2.2 kg/m²) (STUDY B). In addition to blood glucose, serum insulinand breath H₂, also appetite regulatory hormones (PYY, GLP-1), andplasma short chain fatty acids (SCFA) were measured.

In STUDY C, 20 subjects from the cohort in STUDY A were chosen based onthe degree of improvements in the glucose regulation at a standardizedbreakfast in an overnight perspective following ingestion of a barleykernel based product (BB) the previous evening. Included were the 10subjects with most pronounced effects and the 10 subjects with leastpronounced effects of BB on glucose regulation. STUDY C is reportedseparately below.

Materials and Methods

Test Subjects

The inclusion criteria were age between 50-70 years, BMI normal toslightly overweight (BMI 18-28 kg/m²), fasting plasma glucose value ≦6.1mmol/L, non-smoker, overall healthy, and no known metabolic disorders orfood allergies. Anti-hypertensive medications and prescription-freepainkillers without any anti-inflammatory action were accepted. Thestudies were approved by the Regional Ethical Review Board in Lund,Sweden (Reference 2010/457).

STUDY A: Healthy volunteers, 6 men and 33 women aged 64.5±5.6 years, andwith normal body mass indices (mean±SD=23.6±2.3 kg/m²) participated inthe study.

STUDY B: Twenty healthy volunteers, 3 men and 17 women, aged 64.1±5.9years and with normal body mass indices (mean±SD 23.5±2.2 kg/m²), wererandomly chosen from the cohort in STUDY A.

Test Meals

The test meals were barley kernel based bread (BB) and white wheat bread(WB; reference meal). Each test product was consumed during threeconsecutive days, separated by two weeks. The quantity of the test andreference product, respectively, was calculated to provide 100 g ofpotentially available starch per day, analysed according to Holm et al.[2] (Table 1). For the two first days the daily intake was divided intothree equal portion sizes to be consumed at approximately 0800, 1400 and2100. On the third day, half of the daily intake (50 g available starch)was equally divided between the 0800—and 1400 meals, and the other halfwas consumed at 2100.

TABLE 1 Composition of the barley kernel bread (BB) and white wheatbread reference (WB) in STUDIES A-C. Avail- In- Total able solubleSoluble Total Products starch RS starch¹ DF DF DF² RS + DF % dry matterWB 77.4 1.92 75.5 3.55 1.38 4.93 6.85 BB 74.3 10.8 63.5 8.83 4.21 13.0423.84 g/day WB 103 2.54 100 4.71 1.83 6.54 9.08 BB 117 17.0 100 13.976.64 20.61 37.61 ¹Calculated by difference; Total starch less RS (3-5).²The DF mentioned in this application does not include physicallyinaccessible- or indigestible starch, i.e. RS.

Standardized Breakfast

A standardised breakfast was consumed after 3 days intervention with BBor WB, respectively, and consisted of 122.9 g WB corresponding to 50 gavailable carbohydrates, and 2.5 dl tap water.

Recipes and Preparation of Test Products.

White Wheat Bread (WB, Reference Meal and Standardized Breakfast);

A WB was baked according to a standardized procedure in a home bakingmachine (Tefal home bread model nr. 573102; Menu choice, program 2[white bread, 1000 g, quick (time2:32)]). The bread was made from 540 gof white wheat flour (Kungsornen Ab, Jarna, Sweden), 360 g water, 4.8 gdry yeast, 4.8 g NaCl (without Iodin). After cooling, the bread wassliced and wrapped in aluminium foil in portions sizes, put into plasticbags and stored in a freezer (−20° C.). The day before consumption thetest persons were instructed to thaw the bread at ambient temperature,still wrapped in aluminium foil and in the plastic bag.

Barley Kernel Based Bread (BB);

A total of 595 g barley kernels was boiled in 520 g water for 12 min andwas then cooled for 30 min in ambient room temperature. All water wasabsorbed into the kernels when cooked. Added to the kernels were 105 gwheat flour, 6 g dry yeast, 5 g salt, and 300 g water. The dough waskneaded for 4 min (Electrolux AKM 3000, N23 N25) and proofed for 30 minin a bowl, followed by another proofing (35 min) in a baking tin. Thebaking tin was covered with aluminium foil and baked in a household ovenat 225° C. withholding a pan of water to maximize steam being present,until inner temperature of the bread reached 96° C. After baking thebread was cooled without the baking tin in wet towels in ambient roomtemperature. After cooling the bread was put in a plastic bag and leftin room temperature during the night. The day after the breads weresliced and wrapped into aluminium foil in portion sizes, put intoplastic bags and stored in a freezer (−20° C.)

Analysis of Total Starch, RS and DF in the Test Products

The test products were analysed with respect to total starch [3], RS[4], and DF [5]. Before analysis of total starch and DF, the breads wereair dried and milled. RS in test products were analysed in products aseaten. Available starch was calculated by subtracting RS from totalstarch (Table 1).

Experimental Procedure.

The study was a randomized (order of the test products) cross-overstudy, meaning that each subject participated in two three daysinterventions, consuming BB or WB, respectively, separated byapproximately 2 weeks. The subjects were encouraged to standardize theirhabitual diet and meal pattern and to avoid alcohol, excessive physicalexercise or food rich in DF during the days of consumption of the testor reference products. Furthermore, they should not have consumedantibiotics or probiotics during the previous 2 weeks and throughout thestudy. After the last test evening meal with barley kernel bread or WB,respectively, the subjects were fasting until a standardized breakfastwas served the next morning. The subjects arrived to the experimentaldepartment at 0730, and an intravenous cannula (BD Venflon, BectonDickinson) was inserted into an antecubital vein to be used for bloodsampling. Fasting blood tests were collected, and satiety and breath H₂registered before consuming the breakfast. The breakfast was consumed at˜0800 and within 13 min. In addition, measures of pre- andpost-breakfast appetite were obtained using a 100 mm Visual AnalogueScale (VAS). During 2.5 h of repeated blood sampling, the subjects weretold to maintain a constant and low degree of physical activity.

Sampling and Analysis of Physiological Variables and Breath Hydrogen inExpired Air.

Finger-prick capillary blood samples were taken for determination ofblood glucose (HemoCue®B-glucose, HemoCue AB, Ängelholm, Sweden). Venousblood samples were collected to determine physiological test markers inserum (s) (s-insulin) and plasma (p) (p-SCFA, p-GLP-1, and p-PYY). Serumand plasma was separated by centrifugation and immediately stored in afreezer (−40° C.) until analysed. Blood collecting tubes intended foranalysis of plasma GLP-1, and PYY were prepared with an inhibitioncocktail consisting of DPPIV (10μ/ml blood) (Millipore, St Charles, USA)and Trasylol® 10 000 KIE/ml Aprotinin (50 μl/ml blood) (Bayer HealthCareAG, Leverkusen, Germany) prior to blood sampling. Tubes containinginhibition cocktail were kept on ice until usage, but maximum for 6days. Commercial Kits based on enzyme-linked immunosorbent assays wereused for determination of S-insulin (Mercodia, Uppsala, Sweden), PYY(3-36 and 1-36), and GLP-1 (active 7-36) (Alpco Diagnostics, Salem USA).SCFA (acetate, propionate, butyrate) were determined using a GC-method[6]. Breath hydrogen (H₂) in expired air was measured as an indicator ofcolonic fermentation activity using a Gastro+(Bedfont EC60 Gastrolyzer,Rochester, England). Faecal samples were collected prior to the dietaryintervention, and at day four, that is after three days consumption oftest—and reference product, respectively. The subjects were instructedto collect faeces from the first defecation that occurred at day fourand the sample was immediately frozen and handed over to theexperimental department within 24 h for continued storage at −80° C.until analysis. A time schedule for determination of the physiologicalparameters is presented in Table 2.

TABLE 2 Time schedule for determination of test markers. Time (minutesafter start of the breakfast) 0 15 30 45 60 90 120 150 P-PYY x x x SCFAx x x P-GLP2 x x x x x H₂ x x x x x x x x Glucose x x x x x x x x P-GLP1x x x x x x S-insulin x x x x x x x Apetite x x x x x x x x sensations

Calculations and Statistical Methods.

GraphPad Prism (version 5, GraphPad Software, San Diegao, Calif., USA)was used for graph plotting. The incremental blood glucose—and seruminsulin areas under the curves (iAUC, (concentrations as a function oftime) were calculated for each subject and test meal, using thetrapezoid model. Incremental peak (iPeak) concentrations were determinedfor glucose and insulin as individual maximum postprandial increase frombaseline. Significant differences in test variables after the differenttest meals were assessed with ANOVA (general linear model), in MINITABStatistical Software (release 16; Minitab, Minitab Inc, State College,Pa.). In the cases of unevenly distributed residuals (tested withAnderson-Darling and considered unevenly distributed when P<0.05), BoxCox transformation were performed on the data prior to the ANOVA.Correlations between parameters were performed using Pearson correlationin MINITAB Statistical Software (release 14; Minitab, Minitab Inc, StateCollege, Pa.). Values of P<0.05 were considered significant. Data areexpressed as means±SEM, values considered significant at P<0.05. Example1: STUDY A: n=39, STUDY B: n=20, STUDY C: n=20.

Results

Study A (N=39)

Blood Glucose and Serum Insulin

Three days consumption of BB significantly improved blood glucoseresponse to the standardised breakfast in terms of postprandial peakconcentrations (iPeak, P<0.01) and iAUC 0-150 min (P<0.01) as comparedto the reference WB (Table 3). In addition, the BB resulted insignificantly reduced insulin iAUC (0-150 min, P<0.001) and insuliniPeak (P<0.001, FIG. 1, Table 3).

TABLE 3 Blood glucose- and serum insulin responses to the standardizedbreakfast after 3 days consumption of barley kernel based bread (BB) orwhite wheat bread (WB), respectively. Glucose (n = 39) Insulin (n = 39)iPeak iAUC 0-150 min iPeak iAUC 0-150 min mmol/L mmol · min/L nmol/Lnmol · min/L WB 3.45 ± 0.14  215 ± 13.6  0.266 ± 0.019   16.2 ± 1.28  BB 2.99 ± 0.14** 181 ± 11.7** 0.230 ± 0.020*** 13.7 ± 1.13*** % Change−13.4 −15.8 −13.6 −15.5 *P < 0.05, **P < 0.01, ***P < 0.001 (differencesbetween WB and BB).

Breath H₂ Excretion

Consumption of the BB significantly increased breath H₂, as compared tothe reference WB, indicative of increased gut fermentation activityafter the BB (P<0.001, Table 4).

TABLE 4 Breath H₂ excretion and appetite sensations fasting and afterthe standardized breakfast, following 3 days consumption of barleykernel based bread (BB) or white wheat bread (WB), respectively. H₂Satiety Hunger Fasting value Mean 0-150 min Fasting value Fasting valueN = 38 N = 39 N = 39 N = 39 Ppm mm WB 11.39 ± 2.23 7.54 ± 1.04  31.44 ±4.33 53.77 ± 3.51  BB 26.32 ± 33.17 ± 4.26*** 39.74 ± 43.67 ± 4.52*3.42*** 4.26* % 131 339.92 26.4 −18.78 Change *P < 0.05, ***P < 0.001(differences between WB and BB)

Appetite Sensations

Three days consumption of BB resulted in increased satiety and reducedhunger at fasting the following day, compared with three daysconsumption of WB (P<0.05, Table 4).

Study A: Summary and Conclusions

Intake of a barley kernel based product resulted in improvedpostprandial glucose regulation at 11-14 hours post ingestion of thelast portion. In addition the barley kernel based product increasedsatiety and reduced hunger sensation in the same time perspective. Inparallel, breath hydrogen excretion increased, indicative of anincreased colonic fermentation activity. The results suggest that thebeneficial effects obtained after the barley kernel based product aremediated by increased gut fermentation, and activation of specificmicrobiota, of the intrinsic DF and RS, present in barley kernel basedproducts.

STUDY B (additional test markers in n=20, randomly chosen from thecohort in STUDY A)

Blood Glucose and Serum Insulin Response

Three days consumption of BB beneficially affected the blood glucoseresponse at the following standardized breakfast in terms of lower iPeak(P<0.05) and iAUC 0-150 min (P<0.05), as compared to three daysconsumption of WB. In addition, s-insulin iAUC (0-150 min) and insuliniPeak were significantly lowered (P<0.01 and P<0.05, respectively) (FIG.2).

Markers of Gut Fermentation: Breath H₂ and SCFA

Three days consumption of BB resulted in significantly increased breathH₂ compared to the reference meal WB (P<0.01, FIG. 3, Table 5). Inaddition, intake of BB significantly increased the concentrations ofs-acetate and concentration of total SCFA (P<0.05, Table 5). Anon-significant increase of circulating s-butyrate was observed after BBcompared to after WB (BB 16.1±0.7; WB 14.2±0.9; P=0.11). The resultsindicate an increased gut fermentation activity after the BB.

TABLE 5 Concentrations of SCFA in plasma at fasting, following 3 daysconsumption of barley kernel based bread (BB) or white wheat bread (WB),respectively. Acetate Propionate Butyrate Total SCFA Fasting valueFasting value Fasting value Fasting value N = 19 N = 19 N = 18 N = 19μmol/L μmol/L μmol/L μmol/L WB 145.3 ± 11.5   9.84 ± 0.902 14.23 ±0.864  170.2 ± 12.6 BB 171.5 ± 10.2* 10.45 ± 0.793 16.01 ± 0.706¹ 197.8± 10.3* % 18 6 13 16 Change *P < 0.05, ¹P = 0.11

Gut-Related Hormones

GLP-1

GLP-1: is a hormone that is produced by the L-cells in the gut and thatincreases insulin secretion from the pancreas and increasesinsulin-sensitivity in both alpha cells and beta cells aglucose-dependent manner. GLP-1 also increases beta cells mass, inhibitsacid secretion and gastric emptying in the stomach, and decreases foodintake by increasing satiety in brain.

Consumption of BB test meals resulted in significantly increasedconcentrations of p-GLP-1 at fasting day four after BB (1.6±0.5 pmol/L)compared with after WB (1.0±0.4 pmol/L) (P<0.01).

GLP-2

The concentrations of p-GLP-2 were significantly increased during theexperimental day after three days consumption of BB (mean 0-150 min:3.5±0.5 ng/ml) compared to after WB (mean 0-150 min: 3.1±0.4 ng/ml,P<0.05).

PYY

PYY: is also produced by L-cells and inhibits gastric motility and hasbeen shown to reduce appetite.

A significant main effect of test meals was observed revealing increasedplasma concentrations of PYY (0-120 min) after three days consumption ofBB, compared to three days intake of WB (P<0.05, FIG. 4).

Relationships Between Gut-Related Hormones and SCFA

After three days consumption of WB or BB, plasma concentrations of PYYcorrelated with plasma concentrations of total SCFA (r=0.51, P<0.05 andr=0.57, P=0.01) respectively.

Study B: Summary and Conclusions:

The extended investigations show that, in addition to the resultspresented in STUDY A, the barley kernel based product increased plasmaconcentrations of gut hormones important to appetite—and glucoseregulation in a time perspective of 11-14 hours after consumption. Anincreased gut fermentation activity was determined with increased breathhydrogen excretion and increased plasma concentrations of SCFA. Inaddition, the barley kernel product increased plasma concentrations ofGLP-2, a gut hormone important to gut barrier functions by reducing theintestine wall permeability to e.g. endotoxins.

The study indicates that gut fermentation of intrinsic indigestiblecarbohydrates present in barley kernel based products may constitute amechanism for a promising approach aiming at prevention and/or treatmentof obesity and associated metabolic disorders

STUDY C (n=20, chosen from the cohort in study a based on degree ofimprovements in the individual glucose regulation at the standardisedbreakfast.

From the cohort in STUDY A (n=39, Example 1), 20 subjects (18 women and2 men), aged (mean±SD) 64.9±5.1 years and with body mass indices(mean±SD) of 23.2±2.4 kg/m², were included in further investigations.The subjects were chosen with respect to the efficiency of the barleykernel product to improve the individual glucose regulation, incomparison to the WB. The 10 subjects (8 women and 2 men, 64.0±4.6years, BMI 23.9±2.7 kg/m²) in STUDY A with most pronounced effects of BBwith respect to benefits on glucose regulation were included and denotedas “responders”, and the 10 subjects (10 women, 65.7±5.3 years, BMI22.5±1.7 kg/m²) with least improvements in glucose regulation of BB wereincluded and denoted “non-responders”. The criteria used to defineresponders and non-responders are described below. In the respondersgroup, three days consumption of BB resulted in significantly improvedglucose tolerance (iAUC 0-150 min: 156±20 and 251±26 mmol*min/L after BBand WB, respectively, P<0.0001, FIG. 5) and decreased insulin responseafter the standardised breakfast (iAUC 0-120 min: 10.3±1.5 and 15.3±2.1nmol*min/L after BB and WB, respectively P<0.01, FIG. 6). In contrast,no significant improvements were seen on glucose tolerance (iAUC 0-150min: 173±23 and 191±24 mmol*min/L after BB and WB, respectively, P=0.11)or insulin responses (iAUC 0-120 min: 15.8±1.8 and 16.3±1.5 nmol*min/Lafter BB and WB, respectively, P=0.51) in the non-responders group.

Faecal samples collected prior to the study, and after consumption of BBand WB, respectively, were characterized with respect to the gutmicrobiota (see Examples 2).

Definition of “Responders” and “Non-Responders”

Responders were defined as those 10 subjects of the cohort in STUDY A(n=39) that achieved the most pronounced beneficial effect of the barleykernel product on glucose regulation. In responders the barley kernelproduct resulted in a decreased incremental blood glucose area (iAUC,0-90 min) after the standardized breakfast by at minimum 25% (between−25-(−67)%, mean=−39%), lowered total AUC in the same period of time,and decreased insulin iAUC after the standardized breakfast with atleast 15% (−15-(−69)%, mean=−33%). Several of the remaining 29 subjectshad either a lowered glucose response or a lowered insulin response.However, the 10 subjects with least improvements in glucose and/orinsulin responses were denoted “non-responder”. In the non-respondersgroup (n=10), four subjects achieved no lowering of the glucose—andinsulin responses after barley, three subjects had slightly loweredglucose responses, but no improvements in insulin responses, and threesubjects achieved slightly lowered insulin response after BB, but noimprovement in glucose responses.

Study C: Summary and Conclusions:

The results show individual differences concerning the beneficialefficiency of barley kernels on glucose regulation in an 11-14 hoursperspective. The reason for the individual discrepancies is suggested tobe related to differences in the composition of the gut microflora.

A FOLLOW UP TO STUDY C was executed. The aim was to confirm the resultspreviously obtained on glucose regulation and gut microflora followingconsumption of the BB versus the WB. The study design was similar to thedesign previously described. All subjects in study c (10 responders and10 non-responders) were asked if they were willing to repeat the study.Seven responders (6 women and 1 man, 65.6±4.3 years, bmi 23.5±3.2 kg/m²)and seven non-responders (7 women, 64.7±6.1 years, bmi 21.7±1.2 kg/m²)accepted results.

The results in the follow-up study were consistent with the previouslyobtained results. Consequently, in the responders group (n=7), the BBsignificantly improved glucose tolerance to the standardised breakfast(iAUC 0-150 min: 239±32 and 295±28 mmol*min/L after BB and WB,respectively, P<0.0001, FIG. 7), meanwhile no improvements of the BBwere seen on glucose tolerance in the non-responders group (iAUC 0-150min: 277±20 and 287±36 mmol*min/L after BB and WB, respectively,P=0.81).

Summary and Conclusions, to Follow Up Study:

The results obtained in the follow-up study thus verifies the resultsobtained in the previous studies described above (studies A-C)concerning the beneficial effects on glucose regulation of DF and RS inBB, and indicates an individual component of the metabolic response.

Example 2

Characterisation of Human Gut Microbiota Following Ingestion of WB or BBProducts in Humans; and Inoculation Experiments in Mice for Validationof Causal Relationships

Fecal DNA Extraction, Amplification, Pyrosequencing and Data Analysis.

Genomic DNA was isolated from 100-150 mg of feces per individual usingthe repeated bead beating (RBB) method previously described by Salonenet al [7]. Fecal DNA was quantified using a NanoDrop ND-1000spectrophotometer (Nano-Drop Technologies) and the genomic DNA qualitywas assessed by gel electrophoresis using 1% Agarose-GelRed gel. Analiquot of the genomic DNA was diluted to 10 ng in 1 ul prior to use forPCR.

Amplification of the V1-V2 variable region of the 16 rRNA gene wasperformed using the 27F and 338R primers fused with 454 Titaniumsequencing adapters to assess fecal microbiota diversity. 338R primerscontained unique error-correcting 12-base barcodes that allow formultiple samples to be analyzed in a single sequencing run. Each samplewas amplified in triplicate in a reaction volume of 25 μL containing 1.5U of FastStart Taq DNA Polymerase (Roche), 0.2 μM of each primer and 1ul (˜10 ng) of the extracted genomic DNA. PCR was carried out under thefollowing conditions: initial denaturation for 3 min at 95° C., followedby 25 cycles of denaturation for 20 sec at 95° C., annealing for 30 secat 52° C. and elongation for 60 sec at 72° C., and a final elongationstep for 10 min at 72° C. After PCR the triplicates were combined andthe resulting product was checked for size and purity on 0.8%Agarose-GelRed gel. Then the samples were purified with the NucleoSpinGel and PCR Clean-up kit (Macherey-Nagel, Germany) and quantified usingthe Quant-iT PicoGreen dsDNA kit (Invitrogen, Carlsbad, Calif.).Purified PCR products were diluted to a concentration of 20 ng in 1 μland pooled in equal amounts. The pooled samples were then purified withthe Ampure magnetic purification beads (Agencourt, Danvers, Mass.) toremove shorter amplification fragments. The pooled products weresequenced using the 454 GS FLX titanium chemistry at National GenomicsInfrastructure (Stockholm).

Raw data were quality filtered to remove sequences that were shorterthan 200 nucleotides, longer than 1,000 nucleotides, contained primermismatches, ambiguous bases, uncorrectable barcodes, or homopolymer runsin excess of six bases. Quality filtered reads were trimmed from their454 adapters and barcode sequences and were analysed with the softwarepackage Quantitative Insights Into Microbial Ecology (QIIME) (version1.5.0). The number of reads that assed the quality filter totaled 755963(mean 12599 sequences/sample). The sequencing data were denoised withdenoise_wrapper.py a wrapper available in QIIME.

Sequences were assigned to operational taxonomic units (OTUs) usingUCLUST with a 97% threshold of pairwise identity. The most abundantsequence was picked as representative for each OTU and was taxonomicallyassigned using the Ribosomal Database Project (RDP) Classifier.Representative OTUs were aligned using Pynast and used to build aphylogenetic tree with FastTree, which was used to estimate β-diversityof samples (weighted UniFrac).

Extraction and Quantification of Short Chain Fatty Acids.

120-190 mg of frozen faeces was transferred to a glass tube (16×125 mm)fitted with a screw cap and 100 μl of stock solution of internalstandard ([1-¹³C]acetate and [²H₆]propionate at 1M concentration,[¹³C₄]butyrate at 0.5M concentration, [1-¹³C₁]isobutyrate and[1-¹³C]isovalerate at 0.1M concentration, [1,2-¹³C₂]hexanoate,[¹³C]lactate and [¹³C₄]succinic acid each in 40 mM concentration) wasadded. Prior to extraction the samples were freeze-dried at −50° C. for3 h (yield 28-78 mg/dry weight). After acidification with 50 μl of 37%HCl, the organic acids were extracted (2 ml diethyl ether/extraction; 2cycles). A 500-μl aliquot of the extracted sample was mixed togetherwith 50 μl of N-tert-butyldimethylsilyl-N-methyltrifluoracetamide(MTBSTFA; Sigma) at room temperature. An aliquot (1 μl) of the resultingderivatized material was injected into a gas chromatograph (AgilentTechnologies 7890 A) coupled to a mass spectrometer detector (AgilentTechnologies 5975 C). A linear temperature gradient was used. Theinitial temperature of 65° C. was held for 6 min, increased to 260° C.(15° C./min) and further to 280° C. for 5 min. The injector and transferline temperatures were 250° C. Quantitation was completed in selectedion monitoring acquisition mode by comparison to labeled internalstandards (valerate was compared to [1-¹³C]isovalerate, heptanoate andoctanoate were compared to [1,2-¹³C₂]hexanoate and fumarate was comparedto [¹³C₄]succinic acid). The m/z ratios of monitored ions were asfollows: 117 (acetic acid), 131 (propionic acid), 145 (butyric acid),146 (isovaleric acid), 159 (isovaleric acid and valeric acid, 173(hexanoic acid), 187 (heptanoic acid), 201 (octanoic acid)), 261 (lacticacid), 287 (fumaric acid), 289 (succinic acid), 121 ([²H₂]—and[1-¹³C]acetate), 136 ([²H₅]propionate), 146 ([1-¹³C₁]isobutyrate), 149([¹³C₄]butyrate), 160 ([1-¹³C]isovalerate), 175 ([1,2-¹³C₂]hexanoate),264 ([¹³C]lactate) and 293 ([¹³C₄]succinic acid).

Results Regarding Faecal Microbial Pattern in Humans

Pyrosequencing of 16S rRNA gene barcoded amplicons resulted in 755 963high-quality sequences, with a mean of 12599 sequences (range7018-21116) per sample with similar amount of sequences generated inresponders (R) and non-responders (NR) after each treatment. Firmicutes(˜70%) was the most abundant phylum in each study group followed byBacteroidetes (˜20%). However, no significant differences in relativeabundance of Firmicutes and Bacteroidetes were observed betweenresponders or non-responders or by any treatment. A non-significantincrease in the abundance of Bacteroidtetes was; however, noted in theResponder group on diet supplemented in barley kernel bread (FIG. 8A).

The Bacteroidetes phylum is composed by Prevotella and Bacteroides andinterestingly we found significantly higher relative abundance in theresponder groups of Prevotella (FIG. 8B, Two-way ANOVA, P<0.01).Furthermore, the levels of Prevotella increased after the barley kernelbread consumption in the responder group, which was not observed in thenon-responders.

Moreover, significant increase in the faecal levels of succinate wasmeasured in the responder group on diet supplemented in barley kernelbread (Table 6). Succinate is known to be the major metabolite from thefermentative activities of Prevotella species.

TABLE 6 Fecal concentration of organic acids. Non- Non- Metabolite Re-Re- Re- Non- re- re- umol/g dry sponder sponder sponder respondersponder sponder weight Control WB BB Control WB BB Acetate 92.82 93.4777.58 90.95 100.92 79.51 Propionate 40.08 36.88 35.12 29.02 37.47 27.57Butyrate 38.02 32.41 30.07 28.27 33.49 23.81 Lactate 3.54 12.86 5.374.73 4.89 8.30 Succinate 4.32 2.86 9.32 1.66 1.33 2.43

Innoculation Experiments in Mice

Ten- to twelve-week-old germ-free Swiss Webster male mice wereinoculated with single gavage of 10⁸ CFU Bacteroidetes thetaiotaomicronstrain VPI-5482 (overnight culture in YCFA medium) or Prevotella copristrain DSM18205 (overnight culture in PYG medium) either in isolation ortogether. Both strains have been isolated from human feaces. Both werecultured and transported to the mice facilities in 15 ml Hungate tubes.Mono- and bi-colonized mice were housed in iso-cage system for 14 days.Prior sacrificing of the mice oral glucose tolerance test (OGTT) wasperformed. All mice were fasted 4 h prior OGTT. The colonization densitywas verified using qPCR assays that used species-specific primers. Acontrol group of age-matched, gnotobiotic male Swiss Webster mice werealso fed the same autoclaved chow diet ad libitum.

Mice were fasted for 4 hours and then given oral gavage of 60% D-glucose(3 g/kg body weight). Blood was drawn from the tail vein at 0, 30, 60,90 and 120 minutes and blood glucose levels were measured using aHemoCue® glucometer. Extra blood was collected from the tail vein at 0,15, and 30 minutes for analysis of serum insulin levels using insulinELISA assay (Crystal Chem, Inc.).

Results

To investigate whether Prevotella has impact on improvements in glucosetolerance we mono colonized gnotobiotic mice with human feces-derivedPrevotella strain—Prevotella copri and compared the glucose tolerance ofthose mice with mice mono colonized with B. thetaiotaomicron andbi-colonized with both strains. Colonization with B. thetaiotamicroncaused impaired glucose tolerance compared with mice colonized with P.copri (FIG. 9A-B). Additionally, the levels of serum insulin in the micemono colonized with P. copri were lower at the 15 and 30 min after oralgavage of glucose, in comparison with the mice mono colonized with B.thetaiotaomicron (FIG. 9C). Importantly, B. thetaiotaomicron colonizedmice exhibited improved glucose tolerance when they were co-colonizedwith P. copri. These data suggest that whereas B. thetaiotaomicronimpairs glucose tolerance and that P. copri can prevent this impairment.

Example 3 Examples of Products

A) A beaker including one serving of yoghurt, with barley DF and/or RSand Prevotella (10⁷ CFU or more), hermetically enclosed in the cap andseparated by a membrane.

B) A bottle including one serving of drinking yoghurt, with barley DF,RS and Prevotella (10⁹ CFU) hermetically enclosed in an accompanyingdrinking straw.

C) One serving of barley DF, RS, and Prevotella hermetically enclosed insingle dose packages.

D) A bottle including one serving of fruit beverage, with barley DFand/or RS and Prevotella (10⁹ CFU) hermetically enclosed in anaccompanying drinking straw.

E) A bottle including one serving of fruit beverage, with barley DF andRS and Prevotella (10⁹ CFU) hermetically enclosed in a cap and separatedby a membrane.

F) An emulsified food product containing the encapsulated Prevotella ina matrix with or without the prebiotic carbohydrate component.

A-F) One portion of barley DF and RS and Prevotella (7 g insolublebarley DF, 3.3 g soluble barley DF, 8.5 g RS, and 10⁹ CFU Prevotella).

REFERENCES

-   1. Vidhyalakshmi, R., R. Bhakyaraj, and R. S. Subhasree,    Encapsulation “The Future of Probiotics”—A Review. Advances in    Biological Research 2009. 3(3-4): p. 96-103.-   2. Holm, J., et al., A rapid method for the analysis of starch.    Starch/Stärke, 1986. 38: p. 224-226.-   3. Björck, I. M. E. and M. A. Siljeström, In-vivo and in-vitro    digestability of starch in autoclaved pea and potatoe products.    Journal of the Science of Food and Agriculture, 1992. 58: p.    541-553.-   4. Åkerberg, A. K., et al., An in vitro method, based on chewing, to    predict resistant starch content in foods allows parallel    determination of potentially available starch and dietary fiber. The    Journal of Nutrition, 1998. 128(3): p. 651-60.-   5. Asp, N.-G., et al., Rapid enzymatic assay of insoluble and    soluble dietary fiber. Journal of Agricultural and Food    Chemistry, 1983. 31: p. 476-482.-   6. Brighenti, F., Summary of the conclusion of the working group on    Profibre interlaboratory study on determination of short chain fatty    acids in blood, in Functional properties of non-digestible    carbohydrates, F. Gullion, et al., Editors. 1998, European    Comission, DG XII, Science, Research and Development: Brussels,    Belgium. p. 150-153.-   7. Salonen, A., et al., Comparative analysis of fecal DNA extraction    methods with phylogenetic microarray: effective recovery of    bacterial and archaeal DNA using mechanical cell lysis. J Microbiol    Methods, 2010. 81(2): p. 127-34.

1. A product for use in the treatment of obesity, the metabolicsyndrome, type 2 diabetes, cardiovascular diseases, dementia, alzheimersdisease and inflammatory bowel disease comprising at least one isolatedbacterial strain from the species Prevotellaceae, wherein the strain isselected from the group consisting of Prevotella copri, Prevotellastercorea, Prevotella histicola, Prevotella ruminicola, PrevotellaBryantii 25A and Prevotella distasonis.
 2. The product for use in thetreatment of obesity, the metabolic syndrome, type 2 diabetes,cardiovascular diseases, dementia, alzheimers disease and inflammatorybowel disease according to claim 1, wherein the product comprisingPrevotella copri.
 3. The product for use in the treatment of obesity,the metabolic syndrome, type 2 diabetes, cardiovascular diseases,dementia, alzheimers disease and inflammatory bowel disease according toclaim 1, wherein the product comprising at least one type of dietaryfibre.
 4. The product for use in the treatment of obesity, the metabolicsyndrome, type 2 diabetes, cardiovascular diseases, dementia, alzheimersdisease and inflammatory bowel disease according to claims, claim 3,wherein the product comprises at least one resistant starch.
 5. Theproduct for use in the treatment of obesity, the metabolic syndrome,type 2 diabetes, cardiovascular diseases, dementia, alzheimers diseaseand inflammatory bowel disease according to claim 1, wherein saidbacterial strain is genetically modified.
 6. The product for use in thetreatment of obesity, the metabolic syndrome, type 2 diabetes,cardiovascular diseases, dementia, alzheimers disease and inflammatorybowel disease according to claim 1, wherein said dietary fibres andresistant starch are related to cereals in any form such as barley-,wheat-, lye-, oat-fibre, beta-glucan.
 7. The product for use in thetreatment of obesity, the metabolic syndrome, type 2 diabetes,cardiovascular diseases, dementia, alzheimers disease and inflammatorybowel disease according to claim 6, wherein said dietary fibre is frombarley.
 8. The product for use in the treatment of obesity, themetabolic syndrome, type 2 diabetes, cardiovascular diseases, dementia,alzheimers disease and inflammatory bowel disease according to claim 1,wherein said resistant starch is in any form such as intrinsic to barleykernels, retrograded starch, botanically encapsulated starch, nativeungelatinized starch, cyklo-dextrins or chemically modified starch. 9.The product for use in the treatment of obesity, the metabolic syndrome,type 2 diabetes, cardiovascular diseases, dementia, alzheimers diseaseand inflammatory bowel disease according to claim 1, comprising anadditional bacterial strain, wherein said strain is a succinateproducing strain.
 10. The product for use in the treatment of obesity,the metabolic syndrome, type 2 diabetes, cardiovascular diseases,dementia, alzheimers disease and inflammatory bowel disease according toclaim 1, comprising succinate.
 11. The product for use in the treatmentof obesity, the metabolic syndrome, type 2 diabetes, cardiovasculardiseases, dementia, alzheimers disease and inflammatory bowel diseaseaccording to claim 1, wherein the bacterial strain from the speciesPrevotellaceae is encapsulated or lyophilised.
 12. A product comprisingas the only bacterial strain one isolated bacterial strain from thespecies Prevotellaceae, wherein the strain is selected from the groupconsisting of Prevotella copri, Prevotella stercorea, Prevotellahisticola, Prevotella ruminicola, Prevotella Bryantii 25 or, Prevotelladistasonis.
 13. A product comprising one isolated bacterial strain fromthe species Prevotellaceae and at least one type of dietary fibre.